Nucleotide binding properties of the G protein α subunit Goα were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [35S]GTPγS (guanosine 5′-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified Goα cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of Goα for GTPγS or Mg2+ were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21ras proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTPγS binding but retained the capacity to interact with the βγ subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type Goα increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl- response, suggesting that this mutation resulted in a dominant negative phenotype.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology